Protofibril-binding antibodies and their use in therapeutic and diagnostic methods for Parkinson&#39;s disease, dementia with lewy bodies and other α-synucleinopathies

ABSTRACT

Antibodies and fragments thereof have high affinity for human α-synuclein protofibrils and low binding of α-synuclein monomers, wherein the antibodies or fragments have specified Complementarity Determining Region (CDR) sequences. Compositions comprise such an antibody or fragment and methods of detecting α-synuclein protofibrils use such an antibody or fragment. In further embodiments, methods of preventing, delaying onset of or treating a neurodegenerative disorder with α-synuclein pathology comprise administering such an antibody or fragment, and such an antibody or fragment is used in the manufacture of a pharmaceutical composition for treatment of a neurodegenerative disorder with α-synuclein pathology. Such an antibody or fragment is used in the diagnosis or monitoring of the development of a neurodegenerative disorder with α-synuclein pathology, and in methods for reducing or inhibiting α-synuclein aggregation by administration of such an antibody or fragment.

RELATED APPLICATIONS

The present application is a 371 of PCT/IB2011/050826 filed Feb. 25,2011 and claims priority under 35 U.S.C. 119 of U.S. Applications Nos.61/308,638 filed Feb. 26, 2010 and 61/406,260 filed Oct. 25, 2010.

FIELD OF THE INVENTION

The present invention is directed to antibodies or fragments thereofhaving high affinity for human α-synuclein protofibrils and low bindingof α-synuclein monomers, wherein the antibodies or fragments havespecified Complementarity Determining Region (CDR) sequences. Thepresent invention is also directed to compositions comprising such anantibody or fragment and to methods of detecting α-synucleinprotofibrils using such an antibody or fragment. In further embodiments,the invention is directed to methods of preventing, delaying onset of ortreating a neurodegenerative disorder with α-synuclein pathology byadministering such an antibody or fragment, and to use of such anantibody or fragment in the manufacture of a pharmaceutical compositionfor treatment of a neurodegenerative disorder with α-synucleinpathology. The invention is also directed to use of such an antibody orfragment in the diagnosis or monitoring of the development of aneurodegenerative disorder with α-synuclein pathology, and to methodsfor reducing or inhibiting α-synuclein aggregation by administration ofsuch an antibody or fragment.

BACKGROUND OF THE INVENTION

Parkinson's disease (PD) and dementia with Lewy bodies (DLB) are the twomost prevalent examples of neurodegenerative disorders with α-synucleinbrain pathology. PD is the most common movement disorder and ischaracterized by rigidity, hypokinesia, tremor and postural instability.PD is believed to affect approximately four to six million peopleworldwide. DLB represents 5-15% of all dementia. In addition toforgetfulness and other dementing symptoms that often fluctuate, DLBpatients typically suffer from recurrent falls and visualhallucinations.

Intraneuronal accumulation of α-synuclein either results in theformation of Lewy bodies, round eosinophilic hyaline 10-20 μm largeinclusions, or Lewy neurites, elongated thread-like dystrophic axons anddendrites. In the PD brain, deposition of Lewy bodies and Lewy neuritesare mostly limited to neurons connecting striatum with substantia nigra.These cells are crucial for the execution of movement and posturalfunctions, explaining the nature of PD symptoms. In the DLB brain,widespread depositions of Lewy bodies and Lewy neurites are found bothin midbrain and cortical areas.

Alpha-synuclein is a protein which is mainly found intraneuronally.Within the neuron, α-synuclein is predominantly located presynapticallyand it has therefore been speculated that it plays a role in theregulation of synaptic activity. Three main isoforms of α-synuclein havebeen identified, of which the longest and most common form comprises 140amino acids. This isoform has been used and alpha-synuclein(α-synuclein) related characteristics of antibodies according to theinvention refer to this isoform of α-synuclein.

In addition to α-synuclein, Lewy bodies consist of a wide range ofmolecules, one of which is 4-hydroxy-2-nonenal (HNE), an α,β-unsaturatedhydroxyalkenal (Qin et al., 2007). It has been shown in vitro that HNEcan modify α-synuclein and thereby facilitate α-synucleinoligomerization. In particular, HNE has been shown to increase andstabilize the formation of protofibrils, i.e. soluble larger oligomericforms of α-synuclein (Qin et al., 2007; WO 2009/133521, incorporatedherein by reference).

Oxidative stress has been implicated in a number of neurodegenerativedisorders characterized by the pathological accumulation of misfoldedα-synuclein. Various reactive oxygen species can induce peroxidation oflipids such as cellular membranes or lipoproteins and also result in thegeneration of highly reactive aldehydes from poly-unsaturated fattyacids (Yoritaka et al., 1996).

Brain pathology indicative of Alzheimer's disease (AD), i.e. amyloidplaques and neurofibrillary tangles, are seen in approximately 50% ofcases with DLB. It is unclear whether the existence of parallelpathologies implies two different diseases or just represents a variantof each respective disorder. Sometimes the cases with co-pathology aredescribed as having a Lewy body variant of AD (Hansen et al., 1990).

Recent research has implicated a role of α-synuclein in AD and Down'ssyndrome, as the α-synuclein protein has been demonstrated to accumulatein the limbic region in these disorders (Crews et al., 2009).

HNE reacts and modifies side chains of cysteine, histidine and lysine,substantially altering the structure and physical properties of theseside chains. Hence, HNE can either react with the C-3 carbon or with thealdehyde group or by combinations thereof. Hence, HNE can covalentlymodify proteins, either inter- or intramolecularly.

Genetics of Parkinson's Disease and Dementia with Lewy Bodies

Rare dominantly inherited forms of PD and DLB can be caused by pointmutations or duplications of the α-synuclein gene. The pathogenicmutations A30P and A53T (Kruger et al., 1998) (Polymeropoulos et al.,1998) and duplication of the gene (Chartier-Harlin et al. 2004) havebeen described to cause familial PD, whereas one other α-synucleinmutation, E46K (Zarranz et al., 2004) as well as triplication of theα-synuclein gene (Singleton et al., 2003) have been reported to causeeither PD or DLB.

The pathogenic consequences of the α-synuclein mutations are only partlyunderstood. However, in vitro data have shown that the A30P and A53Tmutations increase the rate of aggregation (Conway et al., 2000). Abroad range of differently composed α-synuclein species (monomers,dimers, oligomers, including protofibrils) are involved in theaggregation process, all of which may have different toxic properties.It is not clear which molecular species exert toxic effects in thebrain. However, recent research suggests that oligomeric forms ofα-synuclein are particularly neurotoxic. Additional evidence for therole of oligomers is given by the observation that certain α-synucleinmutations (A30P and A53T) causing hereditary Parkinson's disease, leadto an increased rate of oligomerization.

It is not completely known how the α-synuclein aggregation cascadebegins. Possibly, an altered conformation of monomeric α-synucleininitiates formation of dimers and trimers, which continue to form highersoluble oligomers, including protofibrils, before these intermediatelysized species are deposited as insoluble fibrils in Lewy bodies. It isalso conceivable that the α-synuclein oligomers, once they are formed,can bind new monomers and/or smaller multimers of α-synuclein and henceaccelerate the fibril formation process. Such seeding effects canpossibly also occur in the extracellular space as recent evidencesuggests that α-synuclein pathology may propagate from neuron to neuronin the diseased brain.

Apart from the neuropathological changes in α-synucleinopathies, levelsof α-synuclein protein are generally increased in affected brain regions(Klucken et al., 2006).

The major pathology in α-synucleinopathies is intracellular, which posesa challenge to the immune therapeutic approach. However, it is likelythat a fraction of actively induced or passively administratedantibodies can bind their target antigens also intraneuronally.Moreover, the identification of α-synuclein in both plasma andcerebrospinal fluid (El-Agnaf et al., 2006) illustrates that the proteinis not exclusively found within neurons. Reducing such extracellularα-synuclein may shift the equilibrium between the intracellular andextracellular protein pools and result also in decreased intracellularα-synuclein. Evidence suggests that α-synuclein in solution canpenetrate lipid bilayers in cellular membranes and thereby becomeinternalized or exported out of the cell. Recent findings demonstratethat α-synuclein exerts toxic effects in the extracellular space, thusproviding a plausible explanation for how α-synuclein pathology spreadsthroughout the brain as the disease progresses. Studies showed that Lewypathology was transmitted to grafted neurons in transplanted PD patients(Li et al. 2008). Furthermore, α-synuclein is transmitted viaendocytocis to neighboring neurons, and cell-to-cell transmission ofα-synuclein aggregates has been linked to neuronal cell death andpathological progression in PD and other α-synucleinopathies (Desplatset al. 2009).

Diagnosis of PD and DLB

There is a need for improved diagnostic tools and methods to identify arisk for a neurodegenerative disease with α-synuclein pathology. Today,no biochemical method can aid the clinician to diagnose the patientclinical symptoms in the early stages of the disease, before substantialdamage to the brain has already occurred.

The importance of accurate diagnostic assays will become even greater asnew therapeutic possibilities emerge. As of today, only symptomatictreatment (by substituting the loss of active dopamine in the brain) isavailable for PD patients. For DLB, even less therapeutic options areavailable. Nevertheless, clinicians are frequently evaluating possiblebeneficial effects on DLB patients with the standard treatment for AD,i.e. cholinesterase inhibitors. In either way, none of the existingtreatment strategies for α-synucleinopathies are directed against theunderlying disease processes. In addition, there is also a need formonitoring the disease progression and the treatment effect. For areview on different approaches aimed at altering the progression ofParkinson's disease, see George et al. 2009.

In view of the above-mentioned involvement of α-synuclein in severalneurodegenerative disorders, there is a need for novel treatments thatcan eliminate or reduce the effect of toxic α-synuclein species, as wellas a need for good biomarkers to monitor new interventions and providegood prognostic specificity.

SUMMARY OF THE INVENTION

The present invention is directed to improved antibodies and fragmentsthereof having high affinity for human α-synuclein protofibrils and lowbinding of α-synuclein monomers. The present invention is also directedto compositions comprising such an antibody or fragment and to methodsof detecting α-synuclein protofibrils using such an antibody orfragment. In further embodiments, the invention is directed to methodsof preventing, delaying onset of or treating a neurodegenerativedisorder with α-synuclein pathology by administering such an antibody orfragment, and to use of such an antibody or fragment in manufacture of apharmaceutical composition for treatment of a neurodegenerative disorderwith α-synuclein pathology. The invention is also directed to use ofsuch an antibody or fragment in the diagnosis or monitoring of thedevelopment of a neurodegenerative disorder with α-synuclein pathology,and to methods for reducing or inhibiting α-synuclein aggregation byadministration of such an antibody or fragment.

In one embodiment, the antibody or fragment thereof has high affinityfor human α-synuclein protofibrils and low binding of α-synucleinmonomers, and has three variable heavy (VH) CDR sequences (VH-CDR-1,VH-CDR-2, and VH-CDR-3) and three variable light (VL) CDR sequences(VL-CDR-1, VL-CDR-2, and VL-CDR-3), wherein the six CDR sequences of theantibody or fragment thereof are selected from the following respectivegroups:

-   VH-CDR-1 SEQ ID NOS: 22, 23, 24, 25, 26 or 27-   VH-CDR-2 SEQ ID NOS: 28, 29, 30, 31, 32, 33 or 34-   VH-CDR-3 SEQ ID NOS: 35, 36, 37, 38, 39 or 40-   VL-CDR-1 SEQ ID NOS: 41, 42, 43, 44, 45 or 46-   VL-CDR-2 SEQ ID NOS: 47, 48 or 49-   VL-CDR-3 SEQ ID NOS: 50, 51, 52, 53, 54 or 55.

In another embodiment, the antibody or fragment thereof has highaffinity for human α-synuclein protofibrils and low binding ofα-synuclein monomers, and has three variable heavy (VH) CDR sequences(VH-CDR-1, VH-CDR-2, and VH-CDR-3) and three variable light (VL) CDRsequences (VL-CDR-1, VL-CDR-2, and VL-CDR-3), wherein the six CDRsequences of the antibody or fragment thereof are selected from thefollowing respective groups, and sequences having greater than 70, 80,90, 95 or 98% similarity with any of said sequences of the respectivegroups:

-   VH-CDR-1 SEQ ID NOS: 22, 23, 24, 25, 26 or 27-   VH-CDR-2 SEQ ID NOS: 28, 29, 30, 31, 32, 33 or 34-   VH-CDR-3 SEQ ID NOS: 35, 36, 37, 38, 39 or 40-   VL-CDR-1 SEQ ID NOS: 41, 42, 43, 44, 45 or 46-   VL-CDR-2 SEQ ID NOS: 47, 48 or 49-   VL-CDR-3 SEQ ID NOS: 50, 51, 52, 53, 54 or 55,    and wherein the antibody or fragment thereof binds to an epitope    within the amino acid region 113-140, e.g. 113-131, and in    particular the epitopes 125-131, 121-124, 121-127, 121-131, 113-123    or 136-140, of immobilized linear α-synuclein in a model system    comprising 15-mer alpha-synuclein peptides with 11 amino acids    overlap.

The antibodies, fragments, compositions and methods according to theinvention provide improvements in the diagnosis, monitoring, prevention,delay of onset and/or treatment of neurodegenerative disorders withα-synuclein pathology in individuals having and/or at risk of developingsuch disorders.

Additional aspects, embodiments and advantages of the variousembodiments of the present invention will be more apparent in view ofthe detailed description.

BRIEF DESCRIPTION OF THE DRAWING

The detailed description will be more fully understood in view of theDrawings, in which:

FIG. 1 shows the performance of protofibril specific monoclonalantibodies as determined by a competition ELISA. The assay was performedwith HNE-stabilized α-synuclein protofibrils as described in Example 4.

FIGS. 2A and 2B show the performance of protofibril specific antibodymAb49/G analyzed by a competition ELISA as described in Example 4. FIG.2A shows protofibril specific monoclonal antibody mAb49/G binds withhigh affinity to human α-synuclein protofibrils stabilized by either HNEor ONE. FIG. 2B shows the monoclonal antibody also binds with highaffinity to HNE-stabilized protofibrils of human mutated forms ofα-synuclein, A30P and A53T.

FIGS. 3A-3C show the performance of protofibril specific antibodiesanalyzed by a competition ELISA as described in Example 4. Theprotofibril specific monoclonal antibodies bind with high affinity towild type human α-synuclein protofibrils stabilized by either HNE(PF-HNE) or ONE (PF-ONE). The monoclonal antibodies also bind with highaffinity to HNE-stabilized protofibrils of human mutated forms ofα-synuclein, A30P (A30P-HNE) and A53T (A30P-HNE).

FIGS. 4A and 4B are directed to the quantification of α-synucleinprotofibrils by sandwich ELISA as described in Example 5. FIG. 4A showsa schematic of the protofibril specific antibody mAb49/G used as boththe capturing antibody and the detection antibody. FIG. 4B shows thestandard curve generated with HNE-stabilized α-synuclein protofibrils.Assay performance reached a limit of quantification LOQ=9 pM.

FIGS. 5A and 5B show the results of the analysis of diseased (DLB) andcontrol human brain extracts with α-synuclein protofibril specificsandwich ELISA as described in Example 6.

FIG. 6 shows analysis of brain extracts of control mice (ntg, nontransgenic) and 5 month old mice from the Khale transgenic (tg) mouse PDmodel as described in Example 7. Brain tissue was extracted with trisbuffered saline (TBS) and with TBS in the presence of Triton. Analysiswas performed with α-synuclein protofibril sandwich ELISA as describedin Example 5. Protofibril specific antibody mAb49/G was used as both thecapturing antibody and the detection antibody. In the graph, the y axisrepresents the absorbance at OD450.

FIGS. 7A-7F show immunohistochemical (IHC) analysis of tissues asdescribed in Example 8. FIG. 7A shows 38E2/7 binding of Lewy bodies andneurites in PD substantia nigra and a positive α-α-synuclein control.FIG. 7B shows 38E2/7 binding of Lewy bodies and neurites in DLB cortexand substantia nigra and a positive α-α-synuclein control. FIG. 7C showsvarious antibodies binding Lewy bodies and neurites in DLB cortex andsubstantia nigra and a negative control. FIG. 7D shows variousantibodies binding Lewy bodies and neurites in PD substantia nigra and anegative control. FIG. 7E shows no binding of 38E2/7 in non-diseaserelated substantia nigra and a positive α-α-synuclein control. FIG. 7Fshows a comparison of 38E2/7 binding and a positive α-Aβ control incortex of an Alzheimer's disease patient.

FIGS. 8A and 8B show the immunoprecipitation of human brain extractswith protofibril selective monoclonal antibody 38E2/7 using a brainextraction protocol as described in Example 9.

FIGS. 9A and 9B show fluorescence data measured using an Axiovert200microscope equipped with a FITC epifluorescence filter as described inExample 10. FIG. 9A shows treated cells while FIG. 9B shows datacalculated as relative % decrease in fluorescence intensity compared toantibody untreated alpha-synuclein over expressing cells, which was setto 100%.

The various figures will be more fully understood in view of theExamples set forth below.

DETAILED DESCRIPTION

In a first embodiment, the present invention is directed to improvedantibodies and fragments thereof having high affinity for humanα-synuclein protofibrils and low binding of α-synuclein monomers. In aspecific embodiment, the antibodies are of class IgG or mutationsthereof. Within the present disclosure, the high affinity for humanα-synuclein protofibrils means that the antibodies or fragments exhibita dissociation constant K_(d) less than 10⁻⁷ M for human α-synucleinprotofibrils. As is known in the art, protofibrils are soluble oligomersof α-synuclein. Typical protofibrils have a molecular weight in a rangeof from about 1000 to about 5000 kDa, suitably measured using sizeexclusion chromatography with globular proteins used as references, butthe invention is not limited to such typical protofibrils. In addition,within the present disclosure, the low binding of α-synuclein monomersmeans that the binding of an antibody or fragment according to theinvention to α-synuclein monomers is at least 100 times less than thatto α-synuclein protofibrils. In a specific embodiment, these bindingaffinities are measured according to competition ELISA, for example, asdescribed in Example 4.

The invention further relates to methods and uses of such antibodies andfragments for improvements in preventing, delaying onset of, treating,monitoring and/or diagnosing of neurodegenerative disorders withα-synuclein pathology, including, but not limited to, Parkinson'sdisease (PD), dementia with Lewy bodies (DLB), the Lewy body variant ofAlzheimer's disease, multiple system atrophy, psychosis, schizophrenia,and Creutzfeldt-Jakob disease. In α-synucleinopathies, aggregatedα-synuclein as Lewy bodies and Lewy neurites accumulate in the brainand, in some indications, also in other organs.

Examples of antibodies according to the invention have been developed byclassical hybridoma techniques. The antibodies may be polyclonal ormonoclonal. In a specific embodiment, the antibodies are monoclonal.While the present disclosure refers in many instances to antibodies andfragments thereof, for purposes of convenience, the term “antibody” inthe present disclosure includes fragments thereof, meaning activefragments thereof, i.e. fragments having the same characteristics thatare used for definition of an antibody according to the invention,namely high affinity for α-synuclein oligomers/protofibrils and lowbinding of α-synuclein monomers. The antibodies and fragments thereofexhibit high efficiency in clearance of pathogenic forms of α-synuclein.

The invented antibodies bind aggregated forms, in particularprotofibrils, comprising α-synuclein that is either unmodified orconjugated, for example, conjugated to 4-hydroxy-2-nonenal (HNE) or4-oxo-2-nonenal (ONE), or other α, β-unsaturated hydroxyalkenals, orpoly-unsaturated fatty acids, that stabilize a pathogenicprotofibril/oligomeric α-synuclein epitope. Said epitope or epitopes arepresent on conformationally altered or modified α-synuclein, i.e.α-synuclein protofibrils and oligomers which are present in human brainfrom patients with α-synucleinopathies, such as, but not limited to,Parkinson's disease, DLB, etc. The invented antibodies also bind thepathogenic protofibril/oligomeric structures formed by α-synucleinmutants, e.g. A30P and A53T (Kruger et al., 1998) (Polymeropoulos etal., 1997) that have been described to cause familial PD. Anotherexample of such targets for antibodies of the invention are protofibrilsformed by the mutant α-synuclein E46K, causing PD or DLB.

In one specific embodiment of the invention, monoclonal antibodies areprovided for differentiating, diagnosing, identifying risk fordeveloping, and/or treating α-synucleinopathology related disorders,including, but not limited to, e.g. Parkinson's disease, dementia withLewy bodies, Lewy body variant of Alzheimer's disease, Alzheimer'sdisease, Down's syndrome, multiple system atrophy, psychosis,schizophrenia, Creutzfeldt-Jakob disease and other neurodegenerativedisorders.

The antibodies and fragments of the invention comprise defined aminoacid sequences of the CDR1-3 regions on the variable light (VL) andvariable heavy (VH) chains from antibodies having high affinity forsoluble α-synuclein protofibrils containing the “PD and or DLB diseaseepitope.” In specific embodiments, the CDR regions are combined withmodifications of the Fc region to modulate effector functions such as,but not limited to, Fc receptor binding, complement factor C1q binding,effecting half-life, complement activation and inflammation processes.The constant region of an antibody has many important functions notablybinding Fc-receptors and complement factor C1q. The latter function canbe inactivated to avoid inflammatory reactions.

The inventive antibodies and fragments having high affinity forα-synuclein protofibrils and low binding to α-synuclein monomers havethe following distinct advantages as compared to other knownimmunotherapeutic treatment modalities:

-   1) The inventive antibodies and fragments target and inactivate or    at least reduce disease causing α-synuclein protofibrils, e.g. by    inhibition of oligomerization (see Example 10) or by other    mechanisms.-   2) The high affinity for α-synuclein protofibrils exhibited by the    inventive antibodies and fragments reduces the clinical dose needed    for an effective treatment.-   3) The inventive antibodies and fragments provide a modality for    accurate dosing in elderly patients compared to an active    immunization strategy, such as vaccine.-   4) The low binding to α-synuclein monomers in the    periphery/systemically thus allow more antibodies/fragments to be    available for binding and elimination of α-synuclein oligomeric    forms in the brain.-   5) The antibodies and fragments reduce the risk for inflammatory    side-effects, e.g. meningioencephalitis, by low or no binding to    complement factor C1q.

One aspect of the invention is the discovery of the antibody amino acidsequences of the CDR regions that play an important role for binding ofhuman wild type and mutant α-synuclein protofibrils. Antibodies havingbinding sites (CDR regions) according to the invention are characterizedby high affinity for wild-type human α-synuclein oligomers/protofibrils,for use as therapeutics or diagnostics.

The basic structure of an immunoglobulin (IgG) molecule comprises twoidentical light chains and two identical heavy chains linked together bydisulphide bridges. The light chain, which is either lambda or kappa,has a variable region (VL) and a constant region (CL) of approximately110 amino acid residues each. The heavy chain has a variable region (VH)of about 110 amino acid residues, but a much larger constant region (CH)of 300-400 amino acid residues, comprising CHγ1, CHγ2 and CHγ3 regionsor domains.

The constant region (Fc) activates the complement system and binds to Fcreceptors on macrophages, microglia and neutrophiles, which ingest anddestroy infecting microorganisms or foreign/non-self antigens. Thisfunction is important since it is part of the therapeutic principle ofthe antibody, i.e. Fc receptor mediated microglial phagocytosis andclearance of α-synuclein protofibrils. Clearance of α-synucleinoligomeric intermediates via the lysosomal degradation pathway has beendemonstrated (Lee et al., 2004). This process involvesreceptor-dependent or receptor-independent endocytosis ofantibody/protofibril complexes, followed by fusion with lysosomes wherethe α-synuclein protofibrils are degraded (Masliah et al., 2005).Receptors that have been suggested to control this process include theThy 1.1 receptor and the lipoprotein receptor-related protein (LPR).

Other anti-α-synuclein clearance mechanisms are likely to operate aswell. The clearance of soluble α-synuclein protofibrils is a centralmechanism of the treatment according to the invention. α-synucleinprotofibrils are considered highly neurotoxic, initiating and drivingthe disease process. Clearance of α-synuclein protofibrils in the brainis of significant clinical value. In addition to clearance ofα-synuclein protofibrils, other α-synuclein aggregated forms includingα-synuclein fibrils, will be reduced indirectly via removal of theprecursor forms to α-synuclein fibrils such as α-synuclein protofibrils,dimers, trimers, tetramers and higher oligomeric forms. Differentα-synuclein forms including protofibrils and fibrils, are inequilibrium. Treatment with a high affinity protofibril binding antibodyand clearance of α-synuclein protofibrils by said antibody will alsohave the advantage to indirectly reduce other α-synuclein aggregated oroligomeric forms. Yet another mechanism of action of the antibodieswould be to block or inhibit α-synuclein toxicity by binding to toxicα-synuclein species and prevent their interactions with neurons.

The respective variable regions of the heavy and light chains containthree hyper variable regions called complementarity determining regionsor CDRs. The CDR regions are short stretches of about 7-23, e.g. 13-23,amino acids, located in the VL and VH regions. The six CDRs regions onone “arm” of the antibody form the “pocket” that binds the antigen.Several definitions of CDR-sequences are used in the literature. SEQ IDNOS: 1-21 define the inventive CDR-sequences using a firstidentification system, and the thus identified CDR-sequences are shownin VL and VH in monoclonal antibodies specific for human wild-type andmutant α-synuclein protofibrils in Table 1 (see Example 2) by theunderlined regions. SEQ ID NOS: 22-55 identify the inventiveCDR-sequences using the known Kabat system, and the thus identifiedKabat CDR-sequences are shown in VL and VH in antibodies specific forhuman wild-type and mutant α-synuclein protofibrils in Table 2 (seeExample 2) by the underlined regions. The identification of theinventive CDR-sequences according to Kabat (SEQ ID NOS: 22-55) are usedin the present disclosure.

Thus, in one embodiment, an antibody according to the invention ischaracterized by having the six CDR sequences (VH-CDR-1, VH-CDR-2,VH-CDR-3, VL-CDR-1, VL-CDR-2, and VL-CDR-3) selected from each of thefollowing respective groups of CDR sequences, in any combination.

VH CDR-1 SEQ ID NO: 1 GFTFNTYAM  SEQ ID NO: 2 GFTFSNYAM  SEQ ID NO: 3GFTFSSYAM  SEQ ID NO: 4 GDSFTSGYW  SEQ ID NO: 22 GFTFNTYAMNSEQ ID NO: 23 GFTFSNYAMS SEQ ID NO: 24 GFTFSSYAMS SEQ ID NO: 25GDSFTSGYWN SEQ ID NO: 26 GFSLTSYGVH SEQ ID NO: 27 GFTFTDYYMS VH CDR-2SEQ ID NO: 5 RIRTKSNDYATYYADSVKG SEQ ID NO: 28 RIRTKSNDYATYYADSVSEQ ID NO: 6 TVTSGGSYTYYPDSVRG SEQ ID NO: 29 TVTSGGSYTYYPDSVSEQ ID NO: 7 TISNGGSYTYYPDSVKG SEQ ID NO: 30 TISNGGSYTYYPDSVSEQ ID NO: 8 YIRYSGNTYYNPSLKS SEQ ID NO: 31 YIRYSGNTYYNPSL SEQ ID NO: 32VIWRGGSTDYSAAF SEQ ID NO: 33 TISTGGSYTYYPDSV SEQ ID NO: 34FIRNKANGYTTEYSASV VH CDR-3 SEQ ID NO: 9 (SEQ ID NO: 35) VGYRPYAMDYSEQ ID NO: 10 (SEQ ID NO: 36) QNFGSRGWYFDV SEQ ID NO: 11 (SEQ ID NO: 37)HSDYSGAWFAY SEQ ID NO: 12 (SEQ ID NO: 38) SYYDYDRAWFAY SEQ ID NO: 39LLRSVGGFAD SEQ ID NO: 40 DYGNYAMDY VL CDR-1SEQ ID NO: 13 (SEQ ID NO: 41) RSSQNIVHSNGNTYLESEQ ID NO: 14 (SEQ ID NO: 42) RSSQSIVNSNGNTYLESEQ ID NO: 15 (SEQ ID NO: 43) SASSSVSYMY SEQ ID NO: 16 (SEQ ID NO: 44)RSSQSLVHSNGNTYLH SEQ ID NO: 45 RSSQTIVHNNGNTYLE SEQ ID NO: 46KSSQSLLYSSNQKNYLA VL CDR-2 SEQ ID NO: 17 (SEQ ID NO: 47) KVSNRFSSEQ ID NO: 18 (SEQ ID NO: 48) RTSNLAS SEQ ID NO: 49 WASTRES VL CDR-3SEQ ID NO: 19 (SEQ ID NO: 50) FQGSHVPLT SEQ ID NO: 20 (SEQ ID NO: 51)QQYHSYPYT SEQ ID NO: 21 (SEQ ID NO: 52) SQSTHVPWT SEQ ID NO: 53FQGSHVPFT SEQ ID NO: 54 QQFHSYPYT SEQ ID NO: 55 QQYYSYPYT

One of the antibodies that was initially selected for certaininteresting characteristics was rejected as it did not fulfil thecriteria defining an antibody according to the present invention. Animportant parameter for this rejection was the comparatively short VHCDR-3 sequence with five amino acids exposed by this antibody.Therefore, it is concluded that the VH CDR-3 sequence needs to be morethan 5 amino acids. In specific embodiments, the VH CDR-3 sequence is 9,10, 11 or 12 amino acids.

In specific embodiments, the antibodies and fragments according to theinvention have the six CDR sequences selected from the followingcombinations:

-   SEQ ID NOS: 22, 28, 35, 41, 47 and 50,-   SEQ ID NOS: 23, 29, 36, 42, 47 and 50,-   SEQ ID NOS: 24, 30, 37, 43, 48 and 51,-   SEQ ID NOS: 25, 31, 38, 44, 47 and 52,-   SEQ ID NOS: 26, 32, 39, 45, 47 and 53,-   SEQ ID NOS: 23, 33, 37, 43, 48 and 54, and-   SEQ ID NOS: 27, 34, 40, 46, 49 and 55.

In additional specific embodiments, antibodies providing desirablespecificity for α-synuclein protofibrils while fulfilling otherimportant characteristics defined herein have the six CDR sequences ofthe antibody or fragment selected from the following respective groups:

-   VH CDR-1 SEQ ID NOS: 23, 24, 25 or 26-   VH CDR-2 SEQ ID NOS: 29, 30, 31 or 32-   VH CDR-3 SEQ ID NO: 36-   VL CDR-1 SEQ ID NOS: 42, 43, 44 or 45-   VL CDR-2 SEQ ID NOS: 47 or 48-   VL CDR-3 SEQ ID NOS: 50, 51, 52 or 53    or selected from the following respective groups:-   VH CDR-1 SEQ ID NOS: 23, 24, 25 or 26-   VH CDR-2 SEQ ID NOS: 29, 30, 31 or 32-   VH CDR-3 SEQ ID NO: 37-   VL CDR-1 SEQ ID NOS: 42, 43, 44 or 45-   VL CDR-2 SEQ ID NOS: 47 or 48-   VL CDR-3 SEQ ID NOS: 50, 51, 52 or 53    or selected from the following respective groups:-   VH CDR-1 SEQ ID NOS: 23, 24, 25 or 26-   VH CDR-2 SEQ ID NOS: 29, 30, 31 or 32-   VH CDR-3 SEQ ID NO: 38-   VL CDR-1 SEQ ID NOS: 42, 43, 44 or 45-   VL CDR-2 SEQ ID NOS: 47 or 48-   VL CDR-3 SEQ ID NOS: 50, 51, 52 or 53    or selected from the following respective groups:-   VH CDR-1 SEQ ID NOS: 23, 24, 25 or 26-   VH CDR-2 SEQ ID NOS: 29, 30, 31 or 32-   VH CDR-3 SEQ ID NO: 39-   VL CDR-1 SEQ ID NOS: 42, 43, 44 or 45-   VL CDR-2 SEQ ID NOS: 47 or 48-   VL CDR-3 SEQ ID NOS: 50, 51, 52 or 53.

As noted previously, the α-synuclein protofibril binding antibodies andfragments according to the invention are characterized by high affinityfor the target. The high affinity, expressed as the dissociationconstant K_(d), is less than 10⁻⁷ M. In additional embodiments, thedissociation constant Ki_(d) for human α-synuclein protofibrils is lessthan 10⁻⁸ M, less than 10⁻⁹ M, less than 10⁻¹⁰ M, or even less than10⁻¹¹ M. These antibodies and fragments have the advantage that they canbe administered at lower doses compared to antibodies with affinitiesaround 10⁻⁶ M or higher. This has a significant clinical advantage asthese high affinity antibodies, which can be administered by injection,can be given subcutaneously since only a low amount of the antibody isneeded to achieve efficacy. Administration modalities are not limited tosubcutaneous or intravenous injections. Furthermore, the lower dosesneeded for efficacy will reduce cost of goods for production of theantibody.

In addition to the high affinity of the antibodies for α-synucleinprotofibrils, the antibodies and fragments exhibit low binding toα-synuclein monomers, and optionally low binding to α-synuclein fibrils.As noted above, the low binding to α-synuclein monomers means that thebinding of an antibody or fragment according to the invention toα-synuclein monomers is at least 100 times less than that to α-synucleinprotofibrils. In more specific embodiments, the binding of an antibodyor fragment according to the invention to α-synuclein protofibrils ismore than 500 times or even more than 1000 times greater than that toα-synuclein monomers.

In another embodiment, the antibodies and fragments exhibit low bindingto α-synuclein fibrils. In more specific embodiments, the binding of anantibody or fragment according to the invention to α-synucleinprotofibrils is more than 100 times, more than 500 times, or even morethan 1000 times, greater than that to α-synuclein fibrils.

In yet another embodiment of the invention, the antibodies and fragmentsexhibit low binding to beta amyloid (Aβ) protofibrils (e.g. K_(d)>10⁻⁵M) and beta amyloid monomers (e.g. K_(d)>10⁻⁵ M).

In yet another embodiment of the invention, the antibodies and fragmentsexhibit low binding to β-synuclein monomer, γ-synuclein monomer, IAPP(islet amyloid polypeptide), and/or the Medin polypeptide, e.g. thebinding of the antibodies and fragments is at least 100 times less toone or more of these peptides/proteins than that to the humanα-synuclein protofibrils.

According to another embodiment of the invention, the antibody orfragment according to the present invention can be defined by thebinding in a model system to a linear epitope in α-synuclein within theamino acid (aa) region 113-140, e.g. aa region 113-131, with aa 125-131,121-124, 121-127, 121-131, 113-123 and 136-140 as examples of specificepitopes. In this model system, 15-mer α-synuclein peptides with an 11amino acid sequence overlap are used (see Example 3 below).

According to an additional embodiment of the invention, an antibody orfragment is provided, having high affinity for human α-synucleinprotofibrils and low binding of α-synuclein monomers, and comprising acombination of one CDR-sequence selected from each of the six CDRsequence groups of SEQ ID NOS: 22-27, 28-34, 35-40, 41-46, 47-49 and50-52, and sequences having greater than 70, 80, 90, 95 or 98%similarity with any of said sequences in each respective group. Theantibody or fragment binds to an epitope within the amino acid (aa)region 113-140, e.g. aa region 113-131, and in particular the epitopesaa 125-131, 121-124, 121-127, 121-131, 113-123 or 136-140, ofimmobilized linear α-synuclein in a model system comprising 15-merα-synuclein peptides with 11 amino acids overlap.

According to another specific embodiment of the invention, the highaffinity α-synuclein protofibril binding antibodies can reduce orinhibit α-synuclein aggregation, thereby reducing levels of solubleoligomeric α-synuclein forms in the brain.

According to another specific embodiment of the invention, the highaffinity α-synuclein protofibril binding antibodies can bind α-synucleinoligomers/protofibrils outside the CNS as well, thereby shifting theequilibrium of said α-synuclein forms over the blood brain barrier insuch a way as to lower CNS levels of said α-synuclein forms (drainage).

According to another specific embodiment of the invention, theantibodies are of IgG class, suitable for therapeutic use which can passover the blood brain barrier. The high affinity α-synuclein protofibrilbinding IgG antibodies may be engineered to reduce complement factor C1qbinding to the CH2 domain of IgG1 and reduce complement activation andrisk of inflammation. This modification can be done in several differentways. One way is to make a chimeric antibody where the CHγ2 domain ofthe IgG1 constant region has been deleted and exchanged for thecorresponding domain from IgG4 or part of the domain that confers C1qbinding. It is well established that IgG4 does not bind C1q and hencedoes not activate the complement cascade. To achieve this, the constantregion of the heavy chain (CH) is engineered is such a way as to combinethe high affinity Fc-receptor domain (CH-γ3) on IgG1 with the IgG4domain (CHγ2) which has no binding for the complement factor C1q. Thisnew antibody containing the chimeric constant heavy chain (IgG1:CHγ1,CHγ2:IgG4, CHγ3:IgG1) has the important properties of both efficientclearance of α-synuclein protofibrils through Fc-receptor mediatedphagocytosis and reduced risk for side-effects, i.e., inflammation suchas meningioencephalitis.

Yet another way of reducing the risk of inflammation is to alter theoligosaccharide structure of the antibody which will reduce complementfactor C1q binding and complement activation. Thirty differentstructures of the complex biantennary oligosaccharides at Asn-297 inhuman IgG1 have been described. The absence of CH2 associatedcarbohydrates is believed to cause a conformational change in the“hinge” region of the antibody, reducing interaction efficacies witheffector molecules and loss of complement activation function and C1qbinding.

The modification of a high affinity human α-synuclein protofibrilbinding antibody by site-directed mutagenesis of Asn-297 to any otheramino acid will generate an antibody of retained Fc-receptor bindingwith less C1q binding and hence reduced risk of inflammation, inparticular at the blood brain barrier. An alternative to modify theglycosylation on the antibody is to express the antibody in a cell typewhere the enzyme N-acteylglucosaminyl-transferase I has beeninactivated. This will yield an antibody with altered carbohydratestructure at Asn-297. A structure of Man₅GlcNAc₂, but not limited tothis structure, is formed. This carbohydrate modification will reducecomplement factor C1q binding and inhibit inflammation (Wright et al.1998). Alternatively, aglycosylated protofibril binding antibodies canbe achieved by culturing cells expressing antibodies in the presence oftunicamycin, which inhibits glycosylation. These antibodies will havealtered complement activating activity as well as altered Fc-receptorfunction (Leatherbarrow et al. 1985). Screening of clones expressingantibodies with low complement activation and high Fc-receptor bindingwill generate protofibril binding antibodies that exhibit highFc-mediated clearance of α-synuclein protofibrils and low C1q binding.

In another embodiment, the high affinity human α-synuclein protofibrilbinding antibody is of IgG subclass, e.g. IgG1 or IgG4, where thecomplement factor C1q binding site has been modified, i.e. Pro331>Ser331(Xu et al. 1994), in such a way as to reduce or inhibit binding ofcomplement factor C1q. Such antibodies are particularly suitable foradministration, i.e., for the treatment, prevention or delaying onset ofa neurodegenerative disorder with α-synuclein pathology, in anindividual with such a disorder or at risk of developing such adisorder, for example, but not limited to, an individual having or atrisk of developing PD. The proline residue at position 331 in human IgG1can also be changed to a threonine or glycine or any other polar aminoacid. This modification can be achieved by standard molecular biologytechniques such as site-directed mutagenesis or DNA deletions.

Yet another aspect of the invention is the use of high affinity humanα-synuclein protofibril binding antibodies to specifically determineprotofibril levels in human or animal tissues, for example, incerebrospinal fluid (CSF), blood, urine, saliva, or brain tissue, as adiagnostic tool or biomarker for, or for monitoring, a neurodegenerativedisorder with α-synuclein pathology. Parkinson's disease (PD), dementiawith Lewy bodies (DLB), the Lewy body variant of Alzheimer's disease,multiple system atrophy, psychosis, schizophrenia, and Creutzfeldt-Jakobdisease are exemplary only of such neurodegenerative disorders withα-synuclein pathology. For example, levels of human α-synucleinprotofibrils in CSF or blood of a PD patient are likely to be differentas compared to a matched elderly control group not having Parkinson'sdisease or any other α-synucleinopathy. A person who is developingParkinson's disease or any other α-synucleinopathy is likely to havealtered levels of α-synuclein protofibril levels in CSF or bloodcompared to control subjects. Hence, determination of α-synucleinprotofibril levels in CSF or blood can provide an early diagnosis of thedisease. This is possible to achieve with the new high affinityα-synuclein protofibril binding antibodies according to the inventionand, in a specific embodiment, may be achieved in combination with asandwich ELISA method (see Example 5), where α-synuclein protofibrilshave been determined down to 9 pM level. Interference of otherα-synuclein forms, particularly α-synuclein monomers, and optionallyα-synuclein fibrils and α-synuclein fragments in the assay, isnegligible.

Examples of suitable methods for assaying α-synuclein protofibrils inthese tissues as well as in cell cultures using an anti-α-synucleinprotofibril antibody comprise immunoassays such as ELISA, RIA, Westernblotting or dot blotting. These methods are suitable to follow treatmentefficacy as measured by protofibril reduction in clinical trials and/oras a diagnostic test. Since α-synuclein protofibrils levels are very lowin CSF and blood, the high affinity α-synuclein protofibril bindingantibody of the invention is advantageous for a diagnostic test, forexample, based on an ELISA method, to allow measurement of low levels ofα-synuclein protofibrils.

According to such methods, the antibody or fragment according to theinvention is added to a biological sample comprising or suspected ofcomprising α-synuclein protofibrils, and the presence of a complexformed between α-synuclein protofibril and the antibody or fragment isdetected. The complex may be detected qualitatively, i.e., the presenceof the complex is detected, or quantitatively, i.e., a concentration ofthe complex or a threshold concentration of the complex, may bedetected, as desired.

In additional embodiments, the invention includes the use of the highaffinity protofibril specific antibodies and fragments in imaging fordetection, localization and quantitation of α-synuclein protofibrils inhuman and animal tissues. The antibody or fragment may be labelled witha detectable label, for example, a radioactive ligand such as I¹³¹, C¹⁴,H³ or Gallium⁶⁸, but not limited to these radioisotopes, and contactedwith a sample or administered for detection purposes. Such methods aresuitable as a diagnostic tool for neurodegenerative disorders withα-synuclein pathology, including, but not limited to, Parkinson'sdisease, dementia with Lewy bodies and other α-synuclein relatedneurodegenerative disorders. In a specific embodiment, such methods maybe conducted to monitor the development of an α-synuclein relateddisease in a subject without or under medication or other possibletreatment.

Therefore, in one aspect of the invention the antibodies are added to abiological sample comprising or suspected of comprising α-synucleinprotofibrils, the concentration of the complex formed between saidprotofibril and said antibody is measured for detection and/orquantification of protofibrils in the sample. In specific embodiments,the detection methods include immunoassay and proximity ligation assay.The biological sample may be an in vitro sample taken from a subject aswell as an in vivo liquid volume.

Yet another aspect of the invention is to make the antibody speciesspecific for use in veterinary medicine. The diagnostic methods outlinedare also suitable for veterinary use.

Another aspect of the invention is the humanization of said antibodiesto avoid side-effect, i.e. to avoid an immunoresponse against theantibodies in humans when used as a therapeutic or diagnostic agent.Such humanization techniques are within the ability of one of ordinaryskill in the art.

The pharmaceutical compositions according to the invention comprise anantibody or fragment as described herein, and a pharmaceuticallyacceptable carrier. In a specific embodiment for therapeutic use, thecompositions are physiologically acceptable formulations comprising atherapeutically active amount of an antibody or fragment according tothe invention in a physiological buffer, for example, but not limitedto, PBS, suitable for administration to humans and/or animals. Theantibody or fragment can be freeze dried for better stability. Thefreeze dried formulation may contain any suitable conventionalexcipients, including stabilizers, lyoprotectants, buffers, and thelike, such as, but not limited to, mannitol, for protecting and/orstabilizing the product during and/or after freeze drying and/orsubsequent storage.

Optionally, the antibody formulation may contain an antibacterial agentor other preservative or additive which does not interfere with thefunction or efficacy of the protofibril binding antibody or fragment.

EXAMPLES

The following examples are provided for illustration and are notintended to limit the invention to these specific examples.

Example 1 α-Synuclein Protofibril Antibodies

Immunization/Polyclonal Antibodies

In the immunization scheme, Balb/C mice are utilized. As antigen, HNEstabilized α-synuclein protofibrils are used. These are produced aspreviously described (WO 2009/133521, incorporated herein by reference),with the following exception: a 60:1 ratio between HNE and α-synucleinis used. For immunization, mice are injected with HNE stabilizedα-synuclein protofibrils and adjuvant (e.g. 3-6 times). One boosterinjection containing HNE-modified α-synuclein protofibrils was carriedout prior to the mice being sacrificed. Blood from immunized mice wasanalyzed for reactivity toward α-synuclein protofibrils and α-synucleinmonomers. The specificity of the polyclonal antibody response wasanalyzed by a direct ELISA. In a typical experiment, a flat bottom highbinding 96-well polystyrene microtiter plate is coated with monomericα-synuclein (unmodified or modified with HNE or other aldehydes),protofibrils/oligomeric α-synuclein (unmodified or modified with HNE orother aldehydes) or fibrillar α-synuclein, at a final concentration of400 ng/well. The wells are blocked with 2% BSA, washed with 0.05%Tween-20/PBS and cell media supernatants (undiluted or diluted 1:1 withphosphate-buffered saline) from investigated polyclonal antibodies areadded to the wells as primary antibodies. Alkalinephosphatase-conjugated goat anti-mouse mouse IgG/IgM antibody (PierceBiotechnology, Rockford, Ill., USA) is used as the secondary antibody ata dilution of 1/1000 Immunoreactivity is visualized usingp-nitrophenyl-phosphate (Sigma-Aldrich, MO, USA).

In the serum, antibodies that specifically recognize α-synucleinprotofibrils/oligomers are detected. In addition, antibodies thatrecognize α-synuclein monomers can be found. The negative controlrepresents a non-immunized mouse.

Hybridoma/Monoclonal Antibodies

Mouse B-cell hybridomas were used to produced monoclonal α-synucleinprotofibril binding antibodies. Spleen cells are isolated and ground insterile phosphate-buffered saline (PBS) and centrifuged at 1200×g for 10min to collect a cell-rich pellet. The cells are further washed with PBSand centrifuged at 1200×g for 10 min. The cell pellet is resuspended inDulbecco's minimum essential medium (DMEM, Invitrogen, La Jolla, Calif.,USA) supplemented with 1% antibiotics. Spleen cells are mixed at 1:1ratio with Sp2/0 cells (mouse myeloma cell line) in DMEM. To facilitatecell fusion, 1 ml of polyethylene glycol (Sigma-Aldrich, St. Louis, Mo.,USA) is added to the cell mixture and the reaction is stopped with theaddition of DMEM. Cells are harvested and the pellet is resuspended inDMEM supplemented with 10% (v/v) fetal bovine serum (Cambrex, CharlesCity, Iowa, USA) and also containing 1% (v/v) sodium pyruvate (Cambrex,Charles City, Iowa, USA), 1% (v/v) antibiotics (Sigma-Aldrich, St.Louis, Mo., USA) and 1% (v/v) L-glutamine (Cambrex, Charles City, Iowa,USA), 5% (v/v) BM condition media (Roche Diagnostics Scandinavia,Bromma, Sweden) and 2% (v/v) HAT media supplement (Sigma-Aldrich, St.Louis, Mo., USA). Cells are plated on 96 well cell culturing plates andallowed to rest and grow for 2 weeks.

Hybridoma cell supernatants are analysed in a direct ELISA. In a typicalexperiment, a flat bottom high binding 96 well polystyrene microtiterplate is coated with monomeric α-synuclein (unmodified or modified withHNE or other aldehydes), oligomeric/protofibrillar α-synuclein(unmodified or modified with HNE or other aldehydes) or fibrillarα-synuclein. The wells are blocked with 1% BSA, washed with PBS-Tween 20(0.05%) and cell media supernatants (undiluted or diluted 1:2 or 1:5with PBS-Tween 20 (0.05%)) from investigated hybridoma are added to thewells as primary antibodies. Horse radish peroxidase-conjugatedHRP-coupled goat anti mouse Ig (Southern Biotechnology, prod. No.1010-05) is used as the secondary antibody at a dilution of 1/5000.Immunoreactivity is visualized using K-Blue Aqueous TMB substrate(Neogen Corp. prod. No. 331177).

Example 2 Amino Acid Sequence of Variable Regions of Heavy Chain (VH)and Light Chain (VL/Vkappa) Monoclonal Antibodies Specific forα-Synuclein Protofibrils

The amino acid sequences of the variable regions of heavy chain (VH) andlight chain (VL), including the CDR regions of the antibodies weredetermined by RT PCR of mRNA template, followed by DNA sequencing. Theamino acid sequences of the variable heavy chain region (VH) and thevariable light chain region (VL) for selected antibodies are shown inTable 1. The positions of the CDR regions 1-3 are underlined and shown.The amino acid sequences of the CDR regions form the structural basisfor binding human wild type and mutant α-synuclein protofibrilsconstituting the “pathogenic epitope” of α-synuclein protofibrils.

The amino acid sequences of the CDR regions 1-3 of the respective VL andVH chains for protofibril specific antibodies according to the inventionare shown in Table 1. In Table 2 CDR-sequences of a series of additionalantibodies according to the invention are included.

The combined amino acid sequences of the CDR1-3 regions of the VH and VLchains create the molecular “pocket” which binds human α-synucleinwild-type protofibrils with high affinity and specificity. This “pocket”forms the structural basis of the “PD/DLB epitope”. Variations in theCDR amino acid sequence length are observed in both the VH chain and theVL chain and are compatible with binding to human α-synucleinprotofibrils. A shorter CDR region provides a more restricted threedimensional structure of the binding pocket of the antibody, whereas alonger CDR region is more flexible.

The CDR sequences as shown in Tables 1 and 2 are embodiments of thepresent invention, as are the amino acid sequences in the “mouseframework” regions of the VH and VL chains, i.e., outside the CDRregions, as well as the human VL and VH framework regions forprotofibril specific antibodies as, but not limited to, those.

Other amino acid substitutions in the CDR regions than what are shown inTables 1 and 2 are compatible with high affinity and high specificitybinding to human α-synuclein protofibrils. Where a polar amino acid ispresent in a particular position in a CDR region that particular aminoacid can be substituted by another polar amino acid, with retained orimproved high affinity and specificity binding to α-synucleinprotofibrils. Likewise, if a non-polar or negatively or positivelycharged amino acid is present at a certain position, that amino acid canbe substituted by a similar amino acid from the same polarity group.

As a particular amino acid or amino acids may exchanged in any positionin the CDR regions by functional equivalents that confer substantiallythe same function and structure to the antibody with regard to affinityfor α-synuclein protofibrils, such constructs are of course within thescope of the present invention. In this regard, antibodies and fragmentshaving greater than 70, 80, 90, 95 or 98% similarity with one of thepreviously indicated VH CDR and VL CDR sequences of the respectivegroups, with the maintained epitope binding as described herein, arewithin the scope of the present invention.

TABLE 1Amino acid sequence of variable regions of heavy chain (VH) and light chain (VL/kappa) from fourdifferent monoclonal antibodies specific for human wild-type and mutant α-synuclein protofibrils.VH-BA1: 49/G (SEQ ID NO: 56) EVQLVETGGGLVQPKGSLKLSCATS GFTFNTYAMNWVRQAPGKGLEWVA RIRTKSNDYATYYADSVKG RITISRDDSQSMLYLQMNNLKTEDTAMYYCVR VGYRPYAMDY WGQGTSVTVSS VH-BA2: 38E2/7 (SEQ ID NO: 57)EVQLVESGGDLVKPGGSLKFSCAAS GFTFSNYAM SWVRQTPDKRLEWVA TVTSGGSYTYYPDSVRGRFTISRDNAKNTLYLQLSSLKSEDTAMYFCAR QNFGS RGWYFDV WGAGTTVTVSSVH-BA3: 38F11/2_8 (SEQ ID NO: 58) EVMLVESGGGLVKPGGSLKLSCAAS GFTFSSYAMSWVRQTPEKRLEWVA TISNGGSYTYYPDSVKG RFTISRDNAKNTLYLQMSSLRSEDTAMYYCAR HSDYSGAWFAY WGQGTLVTVSA VH-BA4: 48B11/8 (SEQ ID NO: 59)EVQLQESGPSLVKPSQTLSLTCSVTG DSFTSGYW NWIRKFPGNKLEYMG YIRYSGNTYYNPSLKSRISITRDTSKNQYYLQLISVTTEDTATFYCAR SYYDYD RAWFAY WGQGALVTVSAVkappa-BA1: 49/G (SEQ ID NO: 60) DVLMTQTPLSLPVSLGDQASISCRSSQNIVHSNGNTYLE WYLQKPGQSPTLLIY KVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYC FQGSHVPLT F GAGTKLELKVkappa-BA2: 38E2/7 (SEQ ID NO: 61) DVLMTQTPLSLPVSLGDQASISCRSSQSIVNSNGNTYLE WYLQKPGQSPKLLIY KVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYC FQGSHVPLT F GAGTTLELKVkappa-BA3: 38F11/2_8 (SEQ ID NO: 62) QIVLTQSPAIMSASPGEKVTISC SASSSVSYMYWYQQKPGSSPKPWIY RTSNLAS GVPARFSGSGSGTSYSLTISSMEAEDAATYYC QQYHSYPYTFGGGTKL EIK Vkappa-BA4: 48B11/8 (SEQ ID NO: 63) DVVMTQTPLSLPVSLGDQASISCRSSQSLVHSNGNTYLH WYLQKPGQSPKLLIY KVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFC SQSTHVPWT F GGGTKLEIK Positions of thevarious CDR regions (1-3) are underlined in VL and VH Antibodies BA1-BA4are examples of high affinity protofibril specific antibodies accordingto the invention.

TABLE 2Amino acid sequences of variable regions of heavy chain and light chain from eight different antibodies specific for   human wild-type and mutant α-synuclein protofibrils.

Positions of the various CDR-regions according to the Kabat system aremarked. The antibodies are examples of high affinity protofibrilspecific antibodies according to the invention. The Heavy Chains arerespectively SEQ ID NOS: 64-71 and the Light Chains are respectively SEQID NOS: 72-79.

Example 3 Epitope Mapping of α-Synuclein Protofibril Specific MonoclonalAntibodies

Epitope mappings of the antibodies were performed by immunoblotting onPepSpots membrane. Synthetic peptides spanning the entire sequence(amino acids 1-140) of human α-synuclein were custom synthesized by JPTPeptide Technologies (a subsidiary of Sigma Aldrich, UK) and immobilizedon the PepSpots membrane. The 33 synthesised 15-mer peptides weredesigned with 11 amino acid sequence overlap. The peptides werecovalently bound to a Whatman 50 cellulose membrane (Whatman, England)by the C-terminus and have usually an acetylated N-terminus due to ahigher stability to degradation. The uncharged N-ac better representsthe region in the native antigen then a charged NH3+-group. The resultsare set forth in Table 3.

TABLE 3 Antibody Epitope Amino acid residue numbers of SEQ ID NO: 8049/G YEMPSEE 125-131 38E2/7 DNEAYEM 121-127 38F11/2_8 LEDMPVDPDNE113-123 48B11/8 DNEA 121-124 47E7/3_47 DNEAYEM 121-127 37D2/14LEDMPVDPDNE 113-123 43B9/1_4 YEPEA 136-140 38E10/13_6_4 DNEAYEMPSEE121-131 Human α-synuclein (SEQ ID NO: 80)

Alternatively, epitope mapping of the antibodies were performed byimmunoblotting on PepSpots membrane (Sigma Aldrich) as follows:Synthetic peptides spanning the C-terminal sequence of human α-synucleinfrom amino acid 100 to 140 were custom synthesized and immobilized onthe PepSpots membrane (Sigma Aldrich). The 30 synthesised 10-merpeptides were designed with 9 amino acid overlap. The results are shownin Table 4.

TABLE 4 Amino acid residue Antibody Epitope numbers of SEQ ID NO: 8038E2/7 EAYEMP 123-128 47E7/3_47 NEAY 122-125

Accordingly, the length and precise position of the epitope depends onthe method used for determination.

Example 4 Characterization of High-Affinity Human α-SynucleinProtofibril Binding Monoclonal Antibodies by Competition ELISA

This example shows four antibodies (mAb49/G, mAb38E2/7, mAb38F11/2_(—)8and mAb48B11/8). These antibodies show high affinity to α-synucleinprotofibrils and low cross-reactivity (binding) to α-synuclein monomers,as measured by means of the competition ELISA assay described below.Briefly, the anti-α-synuclein antibody to be tested is allowed tointeract in solution with α-synuclein monomers or protofibrils andthereafter the mix is added to a microtiter plate precoated withα-synuclein protofibrils. If the antibody binds to the antigen in thepre-incubation step, fewer antibodies will bind to the immobilizedantigen on the microtiter plate. Antibody bound to the immobilizedantigen is detected by an alkaline phosphates enzyme (ALP) conjugatedsecondary antibody. The conjugate is incubated with ALP substrate (pNPP)generating a yellow color that can be detected in a microtiter platereader at 405 nm. Consequently, a low OD value reflects a high affinityof the antibody to the antigen in solution.

Specifically, a high-binding ELISA microtiter plate was coated with 100μl/well of 1 μg/ml of α-synuclein protofibril, diluted in 1×PBS, sealedwith adhesive sealer and incubated overnight at +4° C. Then, the coatingsolution was discarded and the residual binding capacity of the platewas blocked by adding 200 μl/well of PBS-Tween 20 (0.05%). The sealedplate was incubated for 60 min at room temperature (R.T.) by shaking at900 rpm.

Meanwhile, peptide solution was prepared in 1×PBS-Tween 20 (0.05%) bydiluting α-synuclein monomers or protofibril to a concentration of 140nM. A 10-step 3× dilution series of α-synuclein monomers andprotofibrils was performed in a volume of 50 μl in a round bottomed, lowprotein binding microtiter plate. To this solution, 50 μl of the testedantibody diluted in PBS-Tween 20 (0.05%) to a concentration of 100 ng/mlwas added and allowed to interact for 60 min at R.T. by shaking at 900rpm. Sub sequentially, these pre-incubated samples were added to thewashed (3× wash) coated high-binding plate and allowed to incubate for15 min at R.T. without shaking. The ELISA plate was then washed toremove unbound antibodies. Bound antibodies were detected with 100 μl ofALP-coupled anti-mouse-IgG (Mabtech, Sweden 3310-4) in diluted 1/1000 inPBS-Tween 20 (0.05%) incubated for 60 min at R.T. by shaking at 900 rpm.

Finally, the ELISA plate was washed to remove unbound antibodies and 100μl of ALP-substrate were added to each well. The plate was kept darkduring incubation at R.T. until a yellow color developed. Absorbancevalues were measured on continuous mode at a wavelength of 405 nm every15 minutes up to 120 minutes. Measurements were used for IC50determinations only if there was linearity between time and absorbance.

IC50 values were calculated as the concentration of either monomers orprotofibrils needed to quench half of the signal in the ELISA. Theconcentration of either α-synuclein monomers or protofibrils used inthis method was determined by means UV-SEC, using a commercialα-synuclein standard as reference (cat. S-1001-1, rPeptide, USA, 0.5 mgas determined with BCA). FIG. 1 shows the absorbance at 450 nm for thefour protofibril specific monoclonal antibodies as determined by thedescribed competition ELISA. The assay was performed with HNE-stabilizedα-synuclein protofibrils.

Further ELISA experiments show that the candidate antibodies display thesame affinity for human α-synuclein protofibrils stabilized by HNE orONE. The antibodies also bind α-synuclein protofibrils/aggregatescomposed of A30P or A53T α-synuclein mutants stabilised by HNE asdemonstrated by means of competition ELISA.

Specifically, FIGS. 2A and 2B show the results of the protofibrilspecific antibody mAb49/G analysed by a competition ELISA. Protofibrilspecific monoclonal antibody mAb49/G binds with high affinity to humanα-synuclein protofibrils stabilized by either HNE or ONE (FIG. 2A). Themonoclonal antibody also binds with high affinity to HNE-stabilizedprotofibrils of human mutated forms of α-synuclein, A30P and A53T (FIG.2B). Aggregation of α-synuclein monomers with ONE generates two distinctpopulations of complexes, as defined by size exclusion chromatography.These two distinct peaks were separately eluted and labelled asFP-ONE_large and FP-ONE_small.

Additionally, FIGS. 3A-3C show the results of additional protofibrilspecific antibodies 38E2/7, 38F11/2_(—)8 and 48B11/8, analysed by acompetition ELISA. The protofibril specific monoclonal antibodies bindwith high affinity to wild type human α-synuclein protofibrilsstabilized by either HNE (PF-HNE) or ONE (PF-ONE). The monoclonalantibodies also bind with high affinity to HNE-stabilized protofibrilsof human mutated forms of alpha synuclein, A30P (A30P-HNE) and A53T(A30P-HNE).

Example 5 Establishment of an α-Synuclein Protofibril Specific SandwichELISA

To enable measurements of α-synuclein protofibrils in biologicalsamples, a sandwich ELISA with mAb49/G as both the capturing antibodyand the detecting antibody was established. This assay measuresα-synuclein protofibrils with limit of quantification LOQ=9 pM (see FIG.4B). Due to uncertainties concerning the size of the α-synucleinprotofibrils used in the standard curve, the concentration in pM isbased on the molecular weight of one α-synuclein monomer (14,000 g/mol).Because the molecular weight of a protofibril has been estimated, bysize exclusion chromatogarphy, to be at least 1,000,000 g/mol, the limitof detection calculated as molar α-synuclein protofibrils could be aslow as 0.13 pM.

α-synuclein protofibrils stabilized by HNE and monomeric α-synucleinwere used to validate the conformation specificity of the ELISA.

An ELISA composed of two identical antibodies requires at least a dimerof a protein to produce a signal. However, a large excess of monomericα-synuclein, which may naturally occur in biological samples, couldinterfere with the α-synuclein protofibril analysis by occupying bindingsites of the capture antibody coat, thus inhibiting the protofibrilsfrom binding. This problem was investigated by adding an increasingexcess of α-synuclein monomer to a fixed concentration of α-synucleinprotofibrils (500 pM, expressed as monomer units) and analyzing it withthe mAb49/G ELISA. A 30 000-fold molar excess of α-synuclein monomer (15μM), as compared to α-synuclein protofibrils (500 pM), did not disturbthe measurements with the mAb49/G sandwich ELISA, as expected sinceα-synuclein monomer binds poorly to the capture antibody.

FIG. 4A shows a schematic illustration of the ELISA binding forquantification of α-synuclein protofibrils by sandwich ELISA. FIG. 4Bshows the standard curve generated with HNE-stabilized α-synucleinprotofibrils. The assay performance reached a limit of quantification,LOQ=9 pM.

Example 6 Analysis of Diseased and Control Human Brain Extracts withα-Synuclein Protofibril Specific Sandwich ELISA

A brain extraction protocol using different detergents was performed,generating three different extracts: TBS extract (FIG. 5A, white bar)comprising extracellular and cytosolic α-synuclein species; Tritonextract (FIG. 5A, striped bar) comprising membrane-associatedα-synuclein species; and SDS extract (FIG. 5B, black bar), comprisingSDS-soluble α-synuclein species. Brain extracts were analyzed from apatient diagnosed with the α-synucleinopathy dementia with lewy bodies(DLB). Brain tissue from cortex and substantia nigra was analyzed. As acontrol, brain tissue from cortex of a subject without detectableimmunohistochemical Lewy body pathology was also analyzed. The sandwichELISA was based on the α-synuclein protofibril specific mAb49/G as boththe capturing antibody and the detection antibody. FIGS. 5A and 5B showthe results of the analysis of diseased and control human brain extractswith α-synuclein protofibril specific sandwich ELISA. The assay allowsfor the quantification of protofibrils at levels >9 pM (limit ofquantification; LOQ=9 pM).

Example 7 Measurement of α-Synuclein Protofibrils in Brain Extract froma PD Transgenic Mouse Model

The presence of α-synuclein protofibrils in cell and mouse models havebeen suggested, though until now there has been no method for directassaying of α-synuclein protofibrils in biological samples. The mAb49/Gsandwich ELISA therefore provides the first opportunity to measureα-synuclein protofibril levels in biological samples and mouse models ofα-synucleinopathies, characterised by the accumulation of aggregatedα-synuclein.

Brain extract samples from transgenic mice over-expressing humanα-synuclein A53T mutant were compared with samples from wild type mice.Brains were homogenized in TBS or TBS+tween and centrifuged prior toanalysis in order to recover the soluble α-synuclein fraction.Measurements of α-synuclein protofibril levels in the TBS-solublefractions of non-transgenic mouse brain homogenates were compared totransgenic mice (Kahle model) (FIG. 6). To ensure that all α-synucleinmeasured in this assay was in a soluble state, all samples werecentrifuged for 5 min at 16000×g before analysis. Levels of α-synucleinprotofibrils were measured in brains from 5 month old transgenic micewith α-synuclein pathology.

FIG. 6 shows the results of the analysis of brain extracts of controlmice (ntg, non transgenic) and 5 month old mice from Khale transgenic(tg) mouse PD model in which the y axis represents the absorbance atOD450.

Example 8 Immunohistochemical (IHC) Analysis of Human Brain Tissue

Cortex and substantia nigra from PD and DLB patients was used to performimmunohistochemical (IHC) analysis as described (Oinas et al. 2010). Ascontrol, cortex and substantia nigra from age-matched non-diseasedpatients was used. Positive antibody control for α-synuclein was a mouseanti-α-synuclein mAb (BD 610787).

Binding to Aβ plaques was evaluated as follows: two consecutive slidesfrom the cortex of an AD patient were treated to display antigens andincubated with either a positive anti-Aβ mAb (mAb158, BioArctic) or witheach one of the candidate antibodies. The bound antibody was detected bya secondary anti-species specific antibody coupled to horse radishperoxidase (HRP). The conjugate was then incubated with the HRPsubstrate DAB, generating a colored precipitate which was detected bylight microscopy. The region in which Aβ plaques were detected by meansof a colored precipitation in the positive control was analyzed in theco respective area in the slides treated with the candidate antibody.The lack of a colored precipitation was evaluated visually andinterpreted as a lack of binding to Aβ plaques by the candidateantibodies.

FIG. 7A shows 38E2/7 binding of Lewy bodies and neurites in PDsubstantia nigra and a positive α-α-synuclein control. FIG. 7B shows38E2/7 binding of Lewy bodies and neurites in DLB cortex and substantianigra and a positive α-α-synuclein control. FIG. 7C shows variousantibodies binding Lewy bodies and neurites in DLB cortex and substantianigra and a negative control. FIG. 7D shows various antibodies bindingLewy bodies and neurites in PD substantia nigra and a negative control.FIG. 7E shows no binding of 38E2/7 in non-disease related substantianigra and a positive α-α-synuclein control. FIG. 7F shows a comparisonof 38E2/7 binding and a positive α-Aβ control in cortex of anAlzheimer's disease patient.

Example 9 Analysis of Human Brain Extracts with Immunoprecipitation (IP)and Western Blot

Immunoprecipitation of human brain extracts with protofibril bindingmonoclonal antibody 38E2/7 was conducted using western blot. A brainextraction protocol using different detergents was performed, generatingfour different extracts: TBS extract, comprising extracellular andcytosolic α-synuclein species; Triton extract, comprisingmembrane-associated α-synuclein species, SDS extract, comprisingSDS-soluble α-synuclein species and FA extract, comprising insolubleα-synuclein. These extracts were immunoprecipitated with magnetic beads,to which antibody 38E2/7 or control antibody 15P were coupled. Antibody15P can bind to α-synuclein protofibrils and monomers equally well andis expected to pull down all species presents. FIG. 8A shows SDS extractof substantia nigra of the DLB patient while FIG. 8B shows Tritonextract of substantia nigra of the DLB patient. As seen, in FIGS. 8A and8B, mAb 38E2/7 only captures α-synuclein protofibrils from DLBsubstantia nigra, both in the Triton and in the SDS extracts, whereasmAb15 captures α-synuclein monomers in all extracts.

Example 10 Analysis of α-Synuclein Oligomerization Inhibition

This example shows the antibody mAb49/G inhibits the oligomerization ofα-synuclein monomers using an in vitro method in which neuronal cellsare transfected with 2 vectors, both containing one copy of α-synuclein(aa 1-140) fused with either the N-terminal or the C-terminal fragmentof GFP. Only those cells, in which α-synuclein has oligomerized,bringing both fragments of GFP together, will generate a greenfluorescent color that can be detected with fluorescent microscopy. Thepresence of an antibody that can inhibit and/or disrupt oligomerizationcan be evaluated by comparing the fluorescence in these cultures,compared to a control to which no antibody as been added.

Specifically, H4 neuroglioma cells were transfected with equimolarratios of DNA-constructs containing either α-synuclein (aa 1-140) fusedwith a N-terminal fragment of Green Fluorescent protein (GFP) (aa 1-155)or α-synuclein (aa 1-140) fused with a C-terminal fragment of GFP (aa156-238) using the FuGENE 6 transfection reagent (Roche Diagnostics,Basel, Switzerland). Simultaneously, a control anti-α-synucleinmonoclonal antibody (mAb5C2, Santa Cruz Bio) and mAb49/G wereextracellularly added to the cells with a final concentration of 1μg/ml. The cells were incubated for 24 hours in 37° C. in 5% CO₂. After24 hours, the cells were moved to 30° C. for complete reconstitution ofthe GFP-fluorophore and incubated for an additional time of 24 hours.Fluorescence was measured using an Axiovert200 microscope equipped witha FITC epifluorescence filter. All data was calculated as relative %fluorescence intensity compared to antibody untreated α-synuclein overexpressing cells, which was set to 100%.

As seen in the FIG. 9A, treatment with mAb49/G showed a significant(*p<0.05) reduction (42% decrease in fluorescence intensity compared tountreated cells) in α-synuclein oligomerization. FIG. 9B shows theresults graphically as a percent fluorescence intensity compared tountreated α-synuclein overexpressing cells, which was set of 100%.

The specific examples and embodiments described herein are exemplaryonly in nature and are not intended to be limiting of the inventiondefined by the claims. Further embodiments and examples, and advantagesthereof, will be apparent to one of ordinary skill in the art in view ofthis specification and are within the scope of the claimed invention.

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What is claimed is:
 1. An isolated antibody or fragment thereof havinghigh affinity for human α-synuclein protofibrils and low binding ofα-synuclein monomers and having a combination of three variable heavy(VH) CDR sequences and three variable light (VL) CDR sequences selectedfrom the following combinations: SEQ ID NOS: 22, 28, 35, 41, 47 and 50,SEQ ID NOS: 23, 29, 36, 42, 47 and 50, SEQ ID NOS: 24, 30, 37, 43, 48and 51, SEQ ID NOS: 25, 31, 38, 44, 47 and 52, SEQ ID NOS: 26, 32, 39,45, 47 and 53, SEQ ID NOS: 23, 33, 37, 43, 48 and 54, and SEQ ID NOS:27, 34, 40, 46, 49 and
 55. 2. The antibody or fragment according toclaim 1, having a CDR sequence combination of SEQ ID NOS: 22, 28, 35,41, 47 and
 50. 3. The antibody or fragment according to claim 1, havinga CDR sequence combination of SEQ ID NOS: 23, 29, 36, 42, 47 and
 50. 4.The antibody or fragment according to claim 1, having a CDR sequencecombination of SEQ ID NOS: 24, 30, 37, 43, 48 and
 51. 5. The antibody orfragment according to claim 1, having a CDR sequence combination of SEQID NOS: 25, 31, 38, 44, 47 and
 52. 6. The antibody or fragment accordingto claim 1, having a CDR sequence combination of SEQ ID NOS: 26, 32, 39,45, 47 and
 53. 7. The antibody or fragment according to claim 1, havinga CDR sequence combination of SEQ ID NOS: 23, 33, 37, 43, 48 and
 54. 8.The antibody or fragment according to claim 1, having a CDR sequencecombination of SEQ ID NOS: 27, 34, 40, 46, 49 and
 55. 9. Apharmaceutical composition comprising an antibody or fragment accordingto claim 1, and a pharmaceutically acceptable carrier.
 10. Thecomposition according to claim 9, wherein the antibody or fragment has aCDR sequence combination of SEQ ID NOS: 22, 28, 35, 41, 47 and
 50. 11.The composition according to claim 9, wherein the antibody or fragmenthas a CDR sequence combination of SEQ ID NOS: 23, 29, 36, 42, 47 and 50.12. The composition according to claim 9, wherein the antibody orfragment has a CDR sequence combination of SEQ ID NOS: 24, 30, 37, 43,48 and
 51. 13. The composition according to claim 9, wherein theantibody or fragment has a CDR sequence combination of SEQ ID NOS: 25,31, 38, 44, 47 and
 52. 14. The composition according to claim 9, whereinthe antibody or fragment has a CDR sequence combination of SEQ ID NOS:26, 32, 39, 45, 47 and
 53. 15. The composition according to claim 9,wherein the antibody or fragment has a CDR sequence combination of SEQID NOS: 23, 33, 37, 43, 48 and
 54. 16. The composition according toclaim 9, wherein the antibody or fragment has a CDR sequence combinationof SEQ ID NOS: 27, 34, 40, 46, 49 and
 55. 17. A method of detectingα-synuclein protofibrils, comprising the steps of adding the antibody orfragment according to claim 1 to a biological sample comprising orsuspected of comprising α-synuclein protofibrils, and detecting thepresence of a complex formed between α-synuclein protofibril and saidantibody or fragment.
 18. The method according to claim 17, wherein theantibody or fragment has a CDR sequence combination of SEQ ID NOS: 22,28, 35, 41, 47 and
 50. 19. The method according to claim 17, wherein theantibody or fragment has a CDR sequence combination of SEQ ID NOS: 23,29, 36, 42, 47 and
 50. 20. The method according to claim 17, wherein theantibody or fragment has a CDR sequence combination of SEQ ID NOS: 24,30, 37, 43, 48 and
 51. 21. The method according to claim 17, wherein theantibody or fragment has a CDR sequence combination of SEQ ID NOS: 25,31, 38, 44, 47 and
 52. 22. The method according to claim 17, wherein theantibody or fragment has a CDR sequence combination of SEQ ID NOS: 26,32, 39, 45, 47 and
 53. 23. The method according to claim 17 wherein theantibody or fragment has a CDR sequence combination of SEQ ID NOS: 23,33, 37, 43, 48 and
 54. 24. The method according to claim 17, wherein theantibody or fragment has a CDR sequence combination of SEQ ID NOS: 27,34, 40, 46, 49 and
 55. 25. Method for reducing the amount of α-synucleinoligomers in a subject in need thereof by administration of an antibodyor fragment according to claim 1 to the subject.